Interpretive Summary: Salmonella Enteritidis (S. Enteritidis or SE) is a zoonotic enteric pathogen of worldwide importance and chickens are a significant reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on S. Typhimirum, the mechanisms involved in SE pathogenicity in human and poultry are poorly understood. In this study, we used transposon mutagenesis to identify SE virulence genes using invasiveness in human intestinal epithelial cells (caco-2) and chicken liver cells (LMH) and survival within chicken macrophages (HD-11) as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of a highly invasive SE strain G1NalR were screened using caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which encode genes known to contribute to enteric infection (eg., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR and rfbMN) in SE as well as other Salmonella serovars. In addition, a strong invasion attenuation was observed for mutants in genes or genomic islands that have not been reported previously (eg., SPI-14, ksgA, SEN0034, SEN2278 and SEN3503) or are absent in S. Typhimurium or in most other Salmonella serovars (eg., pegD, SEN1152, SEN1393, SEN1966). Several mutants with reduced caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells, impaired survival in chicken macrophages and in egg albumen, suggesting that these genes are needed for the systemic infection in chickens and may also contribute to egg contamination. In conclusion, this study has identified several novel genes that perform virulence functions in both human and chicken host. This study will further our understanding of the pathogenesis of SE in both human and chicken host.

Technical Abstract:
Salmonella Enteritidis is the world’s leading cause of food borne salmonellosis and illness in people is linked strongly to its contamination of eggs produced by otherwise healthy appearing hens. Salmonella Enteritidis is noted for generating exceptional strain heterogeneity despite having a clonal population structure. It is important to know which sets of genes are associated with virulence or might contribute to a protective immune response. In this manuscript, the expression of genes in strains that vary in their ability to cause disease was investigated following infection of different human and chicken cell lines. In addition, gene expression that contributed to survival in egg albumen was investigated. Several novel genes were identified that appear to contribute to virulence. This study will further our understanding of why Salmonella Enteritidis persistently threatens the safety of food.